This invention pertains to compositions and methods for long-term contraception or sterilization of mammals.
Compositions that have sometimes been used for long-term contraception include those based upon natural or synthetic steroidal hormones to xe2x80x9ctrickxe2x80x9d the female reproductive tract into a xe2x80x9cfalse pregnancy.xe2x80x9d These steroidal hormones must be administered repeatedly to prevent completion of the estrous cycle and conception. Steroids have side effects that can be potentially dangerous.
P. Olson et al., xe2x80x9cEndocrine Regulation of the Corpus Luteum of the Bitch as a Potential Target for Altering Fertility,xe2x80x9d J. Reprod. Fert. Suppl., vol. 39, pp. 27-40 (1989) discusses the luteal phase and its regulation in bitches. The following discussion appears at page 37: xe2x80x9cSpecific toxins can be linked to an antibody or hormone and carried to a specific target cell (or cells) which is then killed by the toxin. The idea of developing a xe2x80x98magic bulletxe2x80x99 has been discussed for decades but is now gaining renewed recognition as a potential, highly selective method for destroying specific tissues while leaving other tissues unharmed. For many years it was impossible to develop large quantities of antibodies which would react specifically with only single antigenic determinants. However, with,the advent of monoclonal antibodies, this problem has been largely overcome. Antibodies can be developed to specific hormone receptors (such as the LH receptor) and then coupled to a toxin. All cells with LH receptors should then be destroyed. Although various cell types have not been characterized in dog corpora lutea, destruction of any luteal cell type could potentially result in luteolysis if cell types communicate.xe2x80x9d (citations omitted)
P. Olson et al., xe2x80x9cNew Developments in Small Animal Population Control,xe2x80x9d JAVMA, vol. 202, pp. 904-909 (1993) gives an overview of methods for preventing or terminating unwanted pregnancies in small animals. The following discussion appears at page 905: xe2x80x9cTissue-specific cytotoxinsxe2x80x94Permanent contraception in females and males might be achieved by administration of a cytotoxin that is linked to gonadotropin-releasing hormone (GnRH) and that selectively destroys gonadotropin-secreting pituitary cells. Similarly, a cytotoxin linked to antibodies against gonadotropin receptors could be targeted to alter gonadal function. Toxins would need to be carefully targeted to specific cells, yet be safe for all other body tissues.xe2x80x9d (citation omitted).
T. Janaky et al., xe2x80x9cShort Chain Analogs of Luteinizing Hormone-Releasing Hormone Containing Cytotoxic Moieties,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 89, pp. 10203-10207 (1992) discloses the use of certain hexapeptide and heptapeptide analogs of GnRH as carriers for certain alkylating nitrogen mustards, certain anthraquinone derivatives, antimetabolite, and cisplatin-like platinum complex.
S. Sealfon et al., xe2x80x9cMolecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor,xe2x80x9d Endocrine Reviews, vol. 18, pp. 180-205 (1997) provides a review of research concerning the interaction between GnRH and its receptor.
D. Morbeck et al., xe2x80x9cA Receptor Binding Site Identified in the Region 81-95 of the xcex2-Subunit of Human Luteinizing Hormone (LH) and chorionic gonadotropin (hCG),xe2x80x9d Molecular and Cellular Endocrinology, vol. 97, pp. 173-181 (1993) disclosed a fifteen amino acid region of LH and hCG that acted as a receptor binding site. (LH and hCG are homologous hormones that produce similar effects.)
S. Cho et al., xe2x80x9cEvidence for autocrine inhibition of gonadotropin-releasing hormone (GnRH) gene transcription by GnRH in hypothalamic GT1-1 neuronal cells,xe2x80x9d Mol. Brain Res., vol. 50, pp. 51-58 (1997) discloses that neuroendocrine populations of GnRH neurons have high affinity receptors for GnRH and for GnRH analogs.
N. Mores et al., xe2x80x9cActivation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release,xe2x80x9d Endocrinology, vol. 137, pp. 5731-5734 (1996) discloses that LH acts directly on neuroendocrine neurons in the brain See also Z. Lei et al., xe2x80x9cSignaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons,xe2x80x9d Mol. and Cell. Endocrinology, vol. 109, pp. 151-157 (1995).
Conventional targeted toxin therapies have several drawbacks. There is a small window for treatment with a particular targeted toxin (on the order of two weeks) before the recipient""s immune system mounts an antibody response to the targeted toxin. These antibodies will neutralize the toxin; or worse, may result in the deposition of the toxin in reticuloendothelial tissues (e.g., liver, spleen, lymph nodes, lungs, bone marrow), where they may damage otherwise healthy tissue. Aside from this drawback, the toxin must be internalized by the targeted cell and translocated into the cytoplasm to have effect.
U.S. Pat. Nos. 5,378,688; 5,488,036; and 5,492,893 disclose compounds said to be useful in inducing sterility in mammals. The disclosed compounds were generically described as GnRH (or a GnRH analog) conjugated to a toxin. The toxin was preferably linked to the sixth amino acid of the GnRH agonist. The toxin was preferably one with a translocation domain to facilitate uptake into a cell. The inventors noted that conjugation of the GnRH agonist to the toxin xe2x80x9cis necessary because, for the most part, the above toxins, by themselves, are not capable of binding with cell membranes in general. That is to say that applicants have found that it is only when a GnRH analog of the type described herein is linked to a toxin of the type noted above does that toxin become capable of binding to cell membranes . . . xe2x80x9d (E.g., U.S. Pat. No. 5,488,036, col. 7, lines 46-52.) The toxins specifically mentioned appear all to have been metabolic toxins, for example ricin, abrin, modeccin, various plant-derived ribosome-inhibiting proteins, pokeweed antiviral protein, xcex1-amanitin, diphtheria toxin, pseudomonas exotoxin, shiga toxin, melphalan, methotrexate, nitrogen mustard, doxorubicin, and daunomycin. None of these toxins is believed to be toxic due to direct interaction with the cell membrane. In the in vivo experiments reported, the most effective time course was reported to be weekly injections for 4 weeks. (E.g., U.S. Pat. No. 5,488,036, col. 20, lines 46-47.) Because most of the conjugates cited are relatively large compounds, antigenicity could be a problem when such multiple administrations are used. The GnRH analog was preferably linked to the toxin with one of several specified heterobifunctional reagents. The specifications suggest that considerable effort was expended in conjugating the toxin to the GnRH agonist. The toxins must in general be internalized into the target cells to have effect, and do not act on cell membranes; in addition, at least some of these toxins must be secondarily transported from the membrane-bound vesicle into the cytoplasm to interact with ribosomes, mitochondria, or other cellular components.
It has been unexpectedly discovered that amphipathic lytic peptides are ideally suited to use in a ligand/cytotoxin combination to specifically induce sterility or long-term contraception in mammals. The peptides act directly on cell membranes, and need not be internalized. Administering a combination of gonadotropin-releasing hormone (GnRH) (or a GnRH agonist) and a membrane-active lytic peptide produces long-term contraception or sterilization in mammals in vivo. Particularly surprising, sterility results even when the combination is administered to a sexually immature animal: The combination then prevents sexual maturation.
The compounds used in the present invention are relatively small, and will not be antigenic. (Lytic peptides are known not to be very antigenic; and the ligands are not antigenic at all.) The compounds may be administered in a single dose, although they may also be given in two or more closely spaced doses. Lysis of gonadotropes has been observed to be very rapid (on the order of ten minutes.) The two componentsxe2x80x94the ligand and the lytic peptidexe2x80x94may optionally be administered as a fusion peptide, or they may be administered separately, with the ligand administered slightly before the lytic peptide, to activate cells with receptors for the ligand, and thereby make those cells susceptible to lysis by the lytic peptide. If a fusion peptide is used, it has been unexpectedly discovered that a linking moiety is not necessary to join the ligand to the lytic peptide: one may be bonded directly to the other, without the need for any intervening linkage; bonding is preferably performed by bonding one end of the ligand to one end of the peptide, not by bonding to the middle of either. The toxin; the lytic peptide, does not need a translocation domain, and need not be internalized, as it binds to and acts directly on the activated cell membrane to cause lysis.
It is known that the D-amino acid form of GnRH will bind to gonadotropes in the pituitary and to GnRH neurons in the brain. It is also known that the D-amino acid forms of lytic peptides have essentially the same propensity to lyse cell membranes as do the L-amino acid forms. Compounds of the present invention (whether administered as a fusion peptide or separately) may therefore be administered either in L-form or D-form. D-form peptides, although generally more expensive than L-form, have the advantage that they are not degraded by normal enzymatic processes, so that the D-form peptides may therefore be administered orally and generally have a longer, biological half-life. Oral administration of the D-form peptide may be enhanced by linking the peptide/hormone fusion product to a suitable carrier to facilitate uptake by the intestine, for example vitamin B12, following generally the B12-conjugation technique of G. Russell-Jones et al., xe2x80x9cSynthesis of LHRH Antagonists Suitable for Oral Administration via the Vitamin B12 Uptake System,xe2x80x9d Bioconjugate Chem., vol. 6, pp. 34-42 (1995).
GnRH or GnRH analogs (collectively, xe2x80x9cGnRH agonistsxe2x80x9d) may be used in the present invention. It has been reported that substitutions at the 6 and 10 positions of the GnRH decapeptide can produce xe2x80x9csuperagonistsxe2x80x9d having greater binding affinity to the GnRH receptor than does GnRH itself. These xe2x80x9csuperagonistsxe2x80x9d include goserelin, leuprolide, buserelin, and nafarelin. See U.S. Pat. No. 5,488,036.
Without wishing to be bound by this theory, it is believed that the mechanism underlying the invention is as follows: GnRH activates gonadotropic cells in the pituitary gland, as well as neuroendocrine GnRH neurons in the brain. The activated cells have substantially increased susceptibility to lysis by a lytic peptide. The lytic peptide then preferentially destroys (or severely damages) these activated cells. When the gonadotrophic cells in the pituitary are destroyed and are deprived of GnRH from the brain, the pituitary no longer secretes follicle stimulating hormone (FSH) or luteinizing hormone (LH), rendering the mammal temporarily or permanently sterile.
Although the ligand and the lytic peptide may be administered separately, it is preferred to link the two in a single molecule, because such a linkage greatly increases the effective concentration of the lytic peptide in the vicinity of ligand-activated cells. Furthermore, this increase in the effective lytic peptide concentration can obviate the need for activation of the cells, allowing the peptide to be linked to a binding site of a ligand alone, without needing to include the xe2x80x9cremainderxe2x80x9d of a native ligand that would normally be needed for activating the target cells. This linkage may be in either order: for example, GnRH/peptide or peptide/GnRH. Examples are GnRH/hecate (SEQ. ID NO. 3) and hecate/GnRH (SEQ. ID NO. 4). Note that no intermediate linker is necessary, and that the carboxy terminus of one of the two peptides may be bonded directly to the amino terminus of the other. (We have found that the initial pyro-glutamic acid residue of the GnRH or the GnRH portion of a fusion peptide may be substituted with glutamine without substantially changing the activity of the respective peptides. See, e.g., SEQ. ID Nos. 9, 3, and 4.)